Bioluminescence is the phenomenon by which visible light is emitted by living organisms or by a substance derived from them through a variety of chemiluminescent reaction systems. Bioluminescence reactions require three major components: a luciferin, a luciferase and molecular oxygen. However, other components may also be required in some reactions, including cations (Ca++ and Mg++) and cofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyse the oxidation of a substrate, luciferin, and produce an unstable intermediate. Light is emitted when the unstable intermediate decays to its ground state, generating oxyluciferin. There are many different unrelated types of luciferin, although many species from at least seven phyla use the same luciferin, known as coelenterazine. In some animals (e.g. jellyfish) the luciferin/luciferase system can be extracted in the form of a stable “photoprotein” which emits light upon calcium binding. Photoproteins differ from luciferases in that they are stabilized oxygenated intermediate complexes of luciferase and luciferin. Photoproteins are present in many marine coelenterates and allow these organisms to emit light for a variety of purposes including breeding, feeding and defense (1). There are many luminescent organisms, but only seven photoproteins, namely Thalassicolin (2,3), Aequorin (4-6), Mitrocomin (syn. with Halistaurin) (7,8), Clytin (syn. with Phialidin) (8,9), Obelin (2,6,10,11), Mnemiopsin (12,13) and Berovin (12,13) have been isolated so far. All these proteins are complexes formed by an apoprotein, an imidazopyrazine chromophore (coelenterazine) and oxygen. Their structures are highly conserved, especially in the region containing the three calcium binding sites (EF-hand structures). The term “photoprotein” identifies the luciferin-bound polypeptide, which is capable of luminescence, while “apophotoprotein” is used to indicate the protein without luciferin.
The most studied photoproteins are Aequorin, isolated from Aequorea victoria (14) and Obelin, isolated from Obelia longissima (15). The photoprotein may be regenerated from the apophotoprotein by incubation with coelenterazine, molecular oxygen, EDTA and 2-mercaptoethanol or dithiothreitol. Since coelenterazine is the common luminescent substrate used by the photoproteins Aequorin, Mitrocomin, Clytin and Obelin, the light-emitting reaction is likely the same in these four photoproteins (16,17).
The Clytin photoprotein was cloned in 1993 by Inouye et al. (18). To date not much work has been done on this photoprotein. The primary structures of aequorin, mitrocomin, clytin and obelin were aligned and showed very strong amino acid sequence identities. The Ca2+-binding sites of Clytin were also found to be highly conserved (19). It was found that hydrozoan Ca2+-binding photoprotein differs from other Ca2+-binding proteins such as calmodulin and troponin C by a relatively high content of cysteine, histidine, tryptophan, proline and tyrosine residues.
The analysis of the primary structure of clytin shows that it contains 198 aminoacidic residues (aa) and belongs to the family of photoproteins.
Photoproteins are widely used in reporter gene technology to monitor the cellular events associated with signal transduction and gene expression.
The study of cellular events and their regulation requires sensitive, non invasive analytic methods. Photoproteins and in general the use of bioluminescence are excellent reporter systems as they have virtually no background in contrast to fluorescence systems.
Photoproteins are expressed in mammalian cells to monitor calcium changes in response to different stimuli. Intracellular calcium concentrations can be measured by adding the cofactor coelenterazine to mammalian cells expressing the photoprotein and detecting photon emission, which is indicative of intracellular calcium concentration. The use of cells which express both a photoprotein and a receptor involved in the modulation of intracellular calcium concentration provides a valid system for the screening of compounds for their effects on the release of intracellular calcium. High throughput screening assays can also be designed using a photoprotein as reporter system. The sensitivity of the system as well as its high signal to noise ratio allow the use of small assay-volumes. Aequorin is up to now the most used photoprotein for these screening assays.
Calcium flux assays are commonly carried out in HTS format utilizing optical screening apparatuses suited for the simultaneous analysis of a high number of samples and equipped with a luminescence imaging system with a CCD Camera detector. However, one of the most used instruments in HTS is FLIPR® (Fluorometric Imaging Plate Reader, Molecular Devices Corporation, Sunnyvale, Calif., USA) which was developed as a high throughput optical screening tool for cell-based fluorescent assays. The apparatus is equipped with an optical detection device that allows for signal isolation on a cell-monolayer, thereby enhancing sensitivity for cell-based assays. The excitation source can be either an Argon laser or a broadband source as a Xenon lamp.
With a light-tight enclosure, extremely sensitive and fast camera, and true simultaneous on-line liquid dispensing, the FLIPR® system most recent versions (FLIPR3 and FLIPRTETRA) have been made suitable also for luminescence assays, even if with lower sensitivity compared to CCD Camera-based equipments.
For the use of the above described instruments FLIPR®, FLIPR3 and FLIPRTETRA and in general for all instruments with a low sensitivity for luminescence assays, a photoprotein with enhanced light emission is highly desirable.